Benefits of Cardiac Resynchronization Therapy in an Asynchronous Heart Failure Model Induced by Left Bundle Branch Ablation and Rapid Pacing

Summary

The establishment of a chronic asynchronous heart failure (HF) model by rapid pacing combined with left bundle branch ablation is presented. Two-dimensional speckle tracking imaging and aortic velocity time integral are applied to validate this stable HF model with left ventricular asynchrony and the benefits of cardiac resynchronization therapy.

Abstract

It is now well recognized that heart failure (HF) patients with left bundle branch block (LBBB) derive substantial clinical benefits from cardiac resynchronization therapy (CRT), and LBBB has become one of the important predictors for CRT response. The conventional tachypacing-induced HF model has several major limitations, including absence of stable LBBB and rapid reversal of left ventricular (LV) dysfunction after cessation of pacing. Hence, it is essential to establish an optimal model of chronic HF with isolated LBBB for studying CRT benefits. In the present study, a canine model of asynchronous HF induced by left bundle branch (LBB) ablation and 4 weeks of rapid right ventricular (RV) pacing is established. The RV and right atrial (RA) pacing electrodes via the jugular vein approach, together with an epicardial LV pacing electrode, were implanted for CRT performance. Presented here are the detailed protocols of radiofrequency (RF) catheter ablation, pacing leads implantation, and rapid pacing strategy. Intracardiac and surface electrograms during operation were also provided for a better understanding of LBB ablation. Two-dimensional speckle tracking imaging and aortic velocity time integral (aVTI) were acquired to validate the chronic stable HF model with LV asynchrony and CRT benefits. By coordinating ventricular activation and contraction, CRT uniformed the LV mechanical work and restored LV pump function, which was followed by reversal of LV dilation. Moreover, the histopathological study revealed a significant restoration of cardiomyocyte diameter and collagen volume fraction (CVF) after CRT performance, indicating a histologic and cellular reverse remodeling elicited by CRT. In this report, we described a feasible and valid method to develop a chronic asynchronous HF model, which was suitable for studying structural and biologic reverse remodeling following CRT.

Introduction

Advanced chronic HF is a leading cause of mortality for various cardiovascular diseases. A subset of patients with congestive heart failure (CHF) also develop ventricular conduction discoordination that aggravates symptoms and prognosis. CRT, also referred to as biventricular pacing, has been introduced as an alternative therapy for these patients for over 20 years^1,2. Unfortunately, about 20-40% of the patients show poor response to CRT. Since then, many studies have been carried out in order to maximize CRT response³. It is now well recognized that patients with LBBB could benefit more from CRT than those with non-LBBB⁴, since an LBBB pattern causes a larger magnitude of cardiac dyssynchrony due to asymmetry in the freedom of wall movement between septal and lateral walls. Meanwhile recent studies have begun exploring changes in gene expression and molecular remodeling associated with CRT⁵. Accompanying the structural reverse remodeling induced by CRT, cellular and molecular reversion to a normal level is of great interest⁶. Hence, it is essential to establish an optimal model of CHF with isolated LBBB for studying CRT benefits.

Chronic, rapid ventricular pacing was once used to produce CHF in a canine model. RV pacing could undoubtedly produce delayed LV contraction as a model of the LBBB-like contraction pattern. However, this type of functional asynchrony with an intact conduction system may not emulate anatomical LBBB and is not considered an appropriate model for studying CRT performance, the essence of which is to coordinate impaired electrical activation and myocardial contraction. Rapid restoration of LV contractility and partial recovery of LV dimensions after cessation of pacing were also reported⁷.

Experimental studies have induced chronic LBBB by RF ablation to establish asynchronous ventricular contraction⁸. A combination of reduction in global pump function and regional invalid mechanical work could exacerbate CHF by generating cardiac inefficiency as well as cardiac remodeling at the tissue, cellular, and molecular levels. In LBBB hearts, workload is lowest in the septum and highest in the LV lateral wall. As a consequence, cardiac remodeling is most pronounced in the lateral wall⁹. The purpose of the present study is: (i) to advance a stable and chronic HF model with interventricular and intraventricular mechanical asynchrony by means of rapid RV pacing in combination with LBB ablation; (ii) to confirm dyssynchronous HF in our model and CRT benefits in coordinating contraction by two-dimensional speckle tracking echocardiography and aVTI; and (iii) to preliminarily explore cellular reverse remodeling elicited by CRT.

Protocol

Fifteen male beagle dogs (12 to 18 months old, weighing around 10.0-12.0 kg) were purchased and subjected to experiments. All procedures were performed in compliance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (Publication No. 85-23, revised 1996) and were approved by the Animal Care Committee in Zhongshan Hospital, Fudan University. Figure 1 shows the schematic workflow for all protocol steps.

1. Pre-surgery Preparation and Baseline Data Collection

1. Shave the hair of one hindlimb for venipuncture. Establish a venous access through the lateral branch of small saphenous vein of experimental beagle dogs using a venous catheter (22 G, 0.9 mm × 25 mm). Inject sodium pentobarbital (30 mg/kg) slowly via the venous catheter to induce anesthesia, which is confirmed by loss of eyelash reflex. Give additional sodium pentobarbital at the dosage of 10 mg/kg in case of anesthesia recovery during the surgery.
2. Secure the limbs to the operation table with coarse rope and keep the animal in a supine position.
3. Clip the hair of the limb extremities and chest. Paste the limb lead electrodes to limb extremities and the precordial lead electrodes at six designated locations on the chest wall. Record the baseline electrocardiogram (ECG).
4. Echocardiographic assessment
   1. Paste the lead electrodes of the echocardiograph to the limbs of the animal.
   2. Carry out a standard echocardiographic examination. From the conventional apical four chamber (A4C) and apical two chamber (A2C) views, obtain the LV end-diastolic volume (LVEDV), LV end-systolic volume (LVESV), and LV ejection fraction (LVEF) calculated using the biplane Simpson's method.
   3. Assess LV longitudinal strain by two-dimensional speckle tracking imaging. Perform speckle tracking on the A4C, A2C, and apical long axis view (APLAX).
   4. Obtain the longitudinal strain curves from the above three apical views at the basal, mid-ventricular, and apical levels in each wall (A4C: septum and lateral wall; A2C: anterior wall and inferior wall; APLAX: anterior-septum and posterior wall). The software will automatically integrate these data to produce a bull's eye map of 17-segment, including 6 segments at the basal level (septum, lateral wall, anterior wall, inferior wall, anterior-septum, and posterior wall), 6 segments at the mid-ventricular level (septum, lateral wall, anterior wall, inferior wall, anterior-septum, and posterior wall), 4 segments at the apical level (septum, lateral wall, anterior wall, inferior wall), and one apical cap.
   5. Time to peak strain (TTP) is defined as the time interval from the beginning of QRS complex to the lowest point of strain curve, which indicates the maximum longitudinal strain. Calculate the standard deviation of the 17-segment TTP (PSD) to evaluate the LV mechanical synchronism.
   6. Record the transaortic Doppler flow velocities in the apical five chamber view. Measure and average the aVTI in 3-4 consecutive beats.
5. Orotracheal intubation and mechanical ventilation
   1. Gently pull out the tongue and maintain extension of the tongue in preparation for orotracheal intubation. Position the animal in a "sniffing" position.
   2. Slowly advance the curved blade of the laryngoscope until the tip of the blade positioned between the base of the tongue and the epiglottis. Lift the laryngoscope upward to expose the vocal cords. Insert an endotracheal tube into the mouth and pass the tube beyond the vocal cords. Secure the tube to the animal head using adhesive tape.
   3. Auscultate both lungs to confirm proper endotracheal tube placement as evidenced by bilateral and symmetrical breath sounds during positive-pressure ventilation.
   4. Connect the outer end of endotracheal tube to a volume cycled respirator. Start and maintain auxiliary mechanical ventilation with room air. Set the breathing frequency at 8-20 times per min with a tidal volume at 8-15 mL/kg. Set the parameters according to SpO₂ measured by pulse oximetry.

2. Epicardial LV Pacing Electrode Implantation

1. Connect the cardiac defibrillator/monitor lead wires to the skin electrodes, which are attached to the limbs. Pre-medicate the animal with 0.3 g  levofloxacin intravenously guttae.
2. After shaving the hair of the neck and chest, sterilize the anterior thoracic region and left cervical region with iodophor and pave the sterile sheets. 
3. Thoracotomy
   1. Perform the muscle-sparing thoracotomy in a right lateral decubitus position. After administering fentanyl by a continuous rate infusion (0.01 mg/kg/hr) intravenously, incise the skin transversely from the left parasternal line at the fourth intercostal space.
   2. After blunt dissection of the 3 layers of thoracic muscle (pectoralis major, pectoralis minor, intercostals), open the left pleural cavity at the fourth intercostal space (between the 4^th and 5^th ribs) by sharp dissection. Place a rib retractor into the intercostal space. Pack sterile gauze immersed in 0.9% NaCl around the lung lobes to protect the lungs and to keep a clear field of vision.
   3. Carefully incise the lateral pericardium using electrocautery. Open the pericardium to fully expose the LV lateral wall with stay sutures (0-suture).
4. LV pacing electrode implantation
   1. Suture the unipolar LV pacing electrode to the myocardium on the LV lateral wall with one stitch using a 4-0 suture. Make a gentle knot on the suture to prevent lacerating the myocardial tissues.
   2. Connect the terminal metal pin of the pacing lead to a bridging cable to test lead parameters. After satisfactory lead parameters are achieved with the pacing threshold <2.0 V at 0.48 ms and lead impedance <2,000 Ω, slightly pull the electrode lead to guarantee a firm fixation.
5. Remove the stay sutures and carefully examine the surgery area to eliminate active bleeding.
6. Close the pericardium with two stitches using 2-0/T sutures. Remove the stuffed gauze and the rib retractor.
7. Use two pericostal sutures (0-suture) to approximate the 4^th and 5^th ribs. Close the intercostal fascia with several stitches using 2-0/T sutures. Inflate the lungs adequately using an auxiliary balloon via orotracheal intubation before the last suture. Look through the intercostals to confirm normal expansion of the lungs.
8. Reposition the muscle layers back in place with no sutures. The pacing lead penetrates the pericardium, intercostal fascia, and muscle layers successively through the gap between surgery knots. 
9. Incise the skin of the left cervical region and dissect the subcutaneous tissue until reaching the deep fascia using a curved clamp. Build a subcutaneous tunnel above the deep facia from the precordial area to the left cervical region with a straight clamp.
10. Pull the terminal pin of the lead through the tunnel to the left cervical region using a straight clamp. Cover the terminal pin with an insulation sleeve, which is ligated using 2-0/T sutures. Suture the lead around the sleeve to the fascia and locally embed the lead at left side of neck.
11. Close the subcutaneous tissue and skin of both the thoracic and cervical incisions using 0-sutures.
12. Stop the anesthesia induction, when the animal is taking spontaneous breaths, disconnect the endotracheal tube from the ventilator. After the animal recovers from anesthesia, remove the tracheal intubation and venous catheter. Keep the animal under observation till full recovery.
13. Inject intramuscularly 800,000 U of penicillin every 12 h for 2 weeks after operation.

3. RA and RV Pacing Electrodes Implantation

1. Implant RA and RV pacing electrodes 2 weeks after LV electrodes implantation, when the animal recovers from the thoracotomy. Carry out the operation in the cardiac catheterization surgery room equipped with a fluoroscopy apparatus.
2. Induce anesthesia as in step 1.1. Secure the limbs to the operation table and maintain the animal in a supine position. Pre-medicate the animal with 0.3 g  levofloxacin intravenously guttae.
3. Clip the hair of limb extremities. Connect the ECG monitor lead wires to the skin electrodes and paste the skin electrodes to limb extremities. Turn on the ECG monitor and select lead II for intra-procedural monitoring.
4. After shaving the hair of the neck, sterilize the left cervical region with iodophor and drape the sterile sheet. Administer fentanyl by a continuous rate infusion (0.01 mg/kg/hr) intravenously during the whole procedure. 
5. Venous approach
   1. Make a small vertical incision close to the preceding wound on the left side of cervical area. Using blunt dissection, separate the fascia to expose the left external jugular vein. Separate the vein from connective tissues carefully with a mosquito clamp.
   2. Gently pull the vein up using a curved clamp and pass two 2-0/T sutures below the vein. Tie off the distal suture.
   3. Lift the distal suture gently and cut a small hole just at the middle of the two sutures with iris scissors. Using a vein pick, insert passive J-shaped RA lead and active RV lead into the left external jugular vein.
6. RV lead implantation
   1. Once the RV lead has been advanced to the low right atrium or inferior vena cava under fluoroscopy, withdraw the straight stylet from the RV lead. Form a J shape at the distal part of the stylet and insert it again through the RV lead.
   2. With the help of the curved stylet, introduce the lead across the tricuspid valve and into the outflow tract. Slowly withdraw both the lead and the stylet, allowing the lead tip to prolapse toward the RV apex.
   3. Replace the curved stylet with a straight one. Advance the lead towards the apex.
   4. Test the lead parameters with the stylet withdrawn about halfway. Satisfactory parameters include a pacing threshold <1.0 V at 0.48 ms, R-wave amplitude >5.0 mV, and lead impedance <2,000 Ω. Once acceptable electrical parameters are obtained, extend the active helix, remove the stylet, and re-measure the parameters.
7. RA lead implantation
   1. Keeping the RA lead directed toward the high anterior atrium, slowly withdraw the straight stylet, allowing retraction of the preformed J-shaped lead with its tip entering the appendage. A characteristic to-and-fro motion of the electrode with atrial activity may be observed.
   2. Satisfactory parameters include a pacing threshold <1.0V at 0.48 ms, P-wave amplitude >2.0 mV, and lead impedance <2,000 Ω. Similarly, when acceptable parameters are obtained, adjust the lead slack and remove the stylet.
8. After checking on the stability of both leads, tighten the suture proximal to the venotomy. Tie down both leads to the underlying deep fascia with two or three 2-0/T sutures around the suture sleeves. Recheck the electrical parameters and position of both leads under fluoroscopy after suturing.
9. Make a pulse generator pocket near the venous entry and in a plane just above the fascial layer and below the subcutaneous fat. Create the pocket using blunt dissection with a curved clamp. It should be just large enough to accommodate both the generator and redundant leads.
10. Clean and dry the lead pins. Cover the terminal pin of the atrial lead with an insulation sleeve and suture the lead to the floor of the pocket. Insert the ventricular lead into a pacemaker pulse generator and tighten it with the distal connector pin past the set-screws of the generator.
11. Place the generator into the pocket with the redundant leads coiling beneath the device. Tie the generator down to the fascia with a 2-0/T suture through the tie-down hole in the header of the generator. Perform the fluoroscopic examination of the entire system.
12. After checking for hemostasis, close the pocket and superficial fascia in layers using 2-0/T sutures. Finally approximate the skin edges with 0-sutures and program the pacemaker to an OVO mode using a telemetry wand.
13. For the animals of the sham group, implant the LV, RV, and RA leads in similar manner, but with no generator insertion.

4. LBB Ablation

1. Carry out the catheter ablation under the guidance of fluoroscopy immediately after RV and RA lead implantation. Shave the hair of the chest, back, and right inguinal region. Keep the animal in a supine position.
2. Prepare a multi-channel electrophysiological recorder for simultaneous surface and intracardiac electrogram recording, with filter settings of 30-400 kHz (bipolar) or 0.05-500 kHz (unipolar), and a signal amplification of 5,000 fold. Attach the cordless return electrode to the back, and the standard 12-lead electrodes to limbs and chest. Connect all leads to the electrophysiological recorder and record the electrogram at a 100 mm/s sweep speed.
3. Venous and arterial approach
   1. After routine disinfection and draping in the right inguinal region, make a small incision vertically through the skin. Using blunt dissection, separate the fascia to identify the right femoral vein and femoral artery.
   2. Gently pull the femoral vein up and place two stay sutures (2-0/T suture) under the vein. Tie off the vein at the distal end. Perform the same maneuver on the femoral artery.
   3. Slightly pick up the femoral vein and introduce a micropuncture needle proximally into the vein between the two sutures. Hold the needle steady and insert a flexible-tip (floppy J-shaped) guidewire through the needle.
   4. When enough guidewire has been passed into the vein, withdraw the needle and advance a dilator and sheath combination (6-Fr) over the guidewire into the femoral vein. Remove the guidewire and dilator with the sheath remaining for catheter introduction. Tie a loose suture around the vein proximal to the venotomy with the venous sheath in place.
   5. Similarly, insert a 7-Fr sheath into the femoral artery. Deliver a bolus of 100 U/kg saline-diluted heparin into the arterial sheath to prevent clotting.
4. Mapping of right-sided His bundle potential
   1. Advance a 6-Fr steerable quadripolar catheter into the femoral vein via the venous sheath. Connect the end of the catheter to the multi-channel electrophysiological recorder via the catheter input module by cables.
   2. Pass the catheter into the right atrium and across the tricuspid valve until it is clearly in the right ventricle. In a right anterior oblique (RAO) 30° fluoroscopy view, withdraw the catheter across the tricuspid orifice until an atrial potential appears and becomes larger. A slight clockwise torque helps to keep the electrodes in contact with the septum. When the atrial and ventricular potentials are approximately equal in size, a biphasic or triphasic deflection appears between them, representing the right-sided His bundle potential.
5. Left bundle branch potential (LBP) mapping and ablation
   1. Introduce a 7-Fr 4 mm-tip steerable ablation catheter into the femoral artery via the arterial sheath. Connect the end of the ablation catheter to the RF generator and multi-channel electrophysiological recorder by cables.
   2. Pass the arterial catheter retrogradely across the aortic valve and advance to the LV in an RAO 30° view. Deflect the catheter tip toward the interventricular septum. Keep the electrode in close contact with the septum.
   3. In a left anterior oblique (LAO) 45° fluoroscopy view, slowly withdraw the catheter along the septum until the left-sided His bundle potential is recorded between atrial and ventricular electrogram, just below the aortic valve. Then slowly advance the catheter along the septum and manipulate the tip to identify a discrete LBP, which is recorded beneath the aortic valve, typically 1-1.5 cm inferior to the left-sided His bundle recording site.
   4. When the potential-to-ventricular electrogram interval is about 10 ms shorter than the HV interval and an A/V electrogram ratio of <1:10 is observed, the LBP is identified. Usually the LBP to the earliest ventricular electrogram interval (LBP-V) is shorter than 20 ms, which could minimize the risk of complete A-V block.
   5. Once a satisfying LBP position is achieved, commence catheter ablation with an RF generator, delivering 500 kHz unmodulated sine-wave energy (power range 30-40 W). Adjust the power to achieve a target temperature of 60 °C at the electrode-tissue interface. If the temperature does not rise above 50 °C within 15 s, discontinue energy delivery, adjust the catheter tip, and start again.
   6. Monitor the impedance continuously during the energy application. A drop in impedance greater than 6-8 Ω during the energy delivery is considered a sign of good tissue contact and adequate heating.
   7. Typical LBBB is defined by: a prolongation of QRS duration; QRS positive in leads I, II, V5, V6 with notched R wave and negative in leads aVR, V1; and a loss of LBP electrogram. If there is no change in QRS morphology after 10 s, stop the energy delivery and adjust the catheter to find a new LBP target. When a typical LBBB QRS morphology appears on the surface electrogram, continue the energy application for 60-90 s or until a sudden rise in impedance.
   8. Stop the energy delivery immediately in the case of complete (3^rd degree) atrioventricular block or ventricular fibrillation (VF). Implement electrical defibrillation rapidly whenever VF occurs.
   9. When an LBBB QRS morphology is achieved, observe the surface electrogram for a stabilization period of 30 min. If a normal QRS morphology reappears, repeat the above-mentioned ablation procedure.
6. Once the catheter ablation procedure is complete, remove both catheters. Remove the venous and arterial sheath and make tight knots rapidly on the proximal sutures to prevent hemorrhage.
7. After careful examination to preclude active bleeding, close the fascia in layers using 2-0/T sutures. Finally close the skin with 0-sutures.
8. Disconnect all electrodes from the animal and monitor the animal frequently until full recovery from anesthesia. Inject intramuscularly 800,000 U of penicillin every 12 h for 1 week after operation.
9. LBB ablation is not performed for the sham group.

5. Rapid Pacing for HF Induction

1. When the animal recovers from the operation, record the surface ECG again to confirm a permanent LBBB presence 1 week later. Then program the pacemaker to a VVI mode at 260 beats per minute (bpm) using a telemetry wand.
2. Induce anesthesia (as in step 1.1) and program the pacemaker to OVO mode after 4 weeks of rapid pacing.
3. Perform echocardiography to assess LVEF (with reference to step 1.4). If the LVEF decreases below 35%, prepare the animal for CRT performance. If the LVEF is still above 35%, submit the animal to rapid RV pacing again by reprogramming the pacemaker to VVI mode at 260 bpm.
4. Perform echocardiography every 2 weeks until the LVEF is below 35%. Once achieving the LVEF target, terminate rapid RV pacing and prepare for CRT strategy.
5. Animals of sham group are not submitted to rapid pacing.

6. Cardiac Resynchronization Therapy Performance

1. Divide the animals with HF into the control group and CRT group randomly. For the HF control group, let the animal survive another 8 weeks with no intervention. For CRT group, start CRT performance via biventricular pacing.
2. After anesthesia induction (as in step 1.1), keep the animal in a supine position by securing the limbs to an operation table. Pre-medicate the animal with 0.3 g  levofloxacin intravenously guttae. Shave the hair of the neck, sterilize the left cervical region, and drape the sterile sheet.
3. After administering fentanyl by a continuous rate infusion (0.01 mg/kg/hr) intravenously, make a small vertical incision immediately next to the previous wound on the left side of neck. Using blunt dissection, separate the fascia to isolate the pulse generator and pacing leads (including the RV, LV, and RA leads) with no injury.
4. Disengage the terminal pin of RV lead from the generator header by loosening the screws. Carefully cut the sutures tightened on the LV and RA leads and uncap the terminal pins. Clean the pins of all leads by immersing in ethanol and using dry gauze successively.
5. Insert the RA, RV, and LV leads correctly into the header of a CRT pulse generator and tighten the screws. Enlarge the pocket using blunt dissection to be fit for the new generator. Place the generator into the pocket and tie the generator down to the floor of the pocket with a 2-0/T suture.
6. Check the pocket for hemostasis. Close the pocket and superficial fascia in layers using 2-0/T sutures. Then close the skin with 0-sutures.
7. Program the pacemaker to a DDD mode with atrioventricular (AV) delay set to a value of 70 ms and interventricular (VV) delay of 0 ms. Examine the animal frequently until recovery from anesthesia. Inject 800,000 U of penicillin intramuscularly every 12 h for 1 week.
8. After 8 weeks of CRT performance, perform the transthoracic echocardiography again on animals of all groups (as in step 1.4).

7. Sacrifice Animals and Histological Analysis

1. Secure the limbs of the animal to the operation table under general anesthesia. Inject 100 mg/kg of intravenous pentobarbital to perform animal sacrifice. Ensure animal death by absence of heart beat and breathing movement.
2. Incise the skin of the left cervical region. With a combination of sharp and blunt dissection, free the generator and the leads. Disconnect all leads from the generator by loosening the screws.
3. Free the leads from subcutaneous tissue gradually and trace them until the venous entry point. Identify the suture sleeves and cut all retention sutures. Retract the active fixation helix of the RV lead to facilitate removal.
4. Make a transverse incision at the fourth intercostal space from the sternal line to the left midclavicular line. Free the LV lead from the precordial fascia, along the subcutaneous tunnel, until the left cervical region, with a straight clamp.
5. After blunt dissection of the thoracic muscles, open the left pleural cavity. Place a rib retractor into the intercostal space. Open the pericardium completely.
6. Cut the suture on the epicardial LV lead electrode. Separate the electrode from the heart by cutting off a small piece of myocardial tissue wrapping around the electrode.
7. Cut open the right chambers along with the septum. Detach the RA and RV electrodes from myocardium using both sharp and blunt dissection. Usually the electrode tips are encapsulated by fibrous and myocardial tissues. Cut the surrounding tissues from the heart if necessary.
8. Pass a straight stylet to the tip of each lead through the terminal pin. Extract the RV and RA leads from the venous entry and remove the LV lead from the subcutaneous tunnel. If the leads cannot be extricated due to fibrous adhesions, strip away the fibrous adhesions along the leads with blunt dissection.
9. Pull the heart up gently, and clip through the heart tissue close to the aorta to excise the heart. Place the heart in a sterile bowl and rinse it several times with physiological saline. Slice the transmural myocardium from the lateral LV wall for histological analysis.
10. Fix myocardial tissues with buffered formalin, then dehydrate, and embed in paraffin. After cutting into 5 µm thick sections, deparaffinize the samples and stain with hematoxylin and eosin (HE) and Masson trichrome.
11. Measure the cellular diameters from the longitudinal sections stained with HE. Express CVF as percentage of collagen-stained area divided by total tissue area in Masson trichrome-stained sections. Select and count five high-powered fields (400x) randomly for each section. Take digital photographs and analyze using a high-resolution digital image analysis system.

Results

Successful LBB Ablation:

Figure 2 represents a typical surface and intracardiac electrogram in the course of catheter ablation. The mean LBP-V measured is 18.8 ±2.8 ms, which was about 10 ms shorter than the baseline H-V interval (28.8 ±2.6 ms, p <0.01). The QRS duration prolonged from 59.2 ±6.8 ms to 94.2 ±8.6 ms (p <0.01) after LBB ablation. The loss of the LBP electrogram con...

Discussion

Dilated cardiomyopathy constitutes a major cause of CHF, which is characterized by ventricular dilation, systolic dysfunction with reduced LVEF, and abnormalities of diastolic filling¹¹. Since chronic tachycardia-mediated HF is a recognized clinical condition, rapid pacing of either atrium or ventricle for at least 3 to 4 weeks serves as a frequently used animal model to induce CHF¹¹. Hemodynamic changes occur as soon as 24 h after rapid pacing, with continued deterioration...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Closed iv catheter system (0.9mm×25mm)	Becton Dickinson Medical	5264442	Used as venous retention needle
Sodium pentobarbital	Sigma-Aldrich Company	130205	For anesthesia
Pet clipper	Wuhan Shernbao pet supplies Co., Ltd.	PGC-660	For hair shaving
Electrocardiograph	Shanghai photoelectric medical electronic instrument Co., Ltd.	ECG-6511	For electrocardiogram recording
Echocardiograph	GE-Vingmed Ultrasound Company	VIVID E9	For echocardiographic assessment
EchoPAC software	GE healthcare	Version201	Offline analysis
Laryngoscope	Shanghai Medical Instrument Co., Ltd		Orotracheal intubation
Endotracheal tube	SIMS Portex Inc, UK	274093	Orotracheal intubation
Volume cycled respirator	Newport Corporation	C100	Artificial ventilation
HeartStart XL Defibrillator/Monitor	Philips Medical Systems	M4735A	Electrocardiogram monitor during operation
Benzalkonium Bromide Tincture	Shanghai Yunjia Pharmaceutical Co., Ltd.	H31022694	Used for skin disinfection
Rib retractor	Shanghai Medical Instrument Co., Ltd.		For thoracotomy
4-0 suture	Shanghai Pudong Jinhuan Medical Products Co., LTD.	24L1005	Suture of LV epicardial electrode
2-0/T suture	Shanghai Pudong Jinhuan Medical Products Co., LTD.	11M0505	Suture of pacing leads, fascia, vessels, etc.
0-suture	Shanghai Pudong Jinhuan Medical Products Co., LTD.	11P0501	Skin suture
penicillin powder	North China Pharmaceutical Co., Ltd.	F6034105
DSA X-ray machine	Philips	Allura Xper FD10	X-ray for fluoroscopy
LV pacing electrode	Medtronic, Inc.	LBT 4965
RV pacing electrode	St. Jude Medical	Tendril 1888
RA pacing electrode	St. Jude Medical	IsoFlex 1642T
Pacemaker pulse generator	Medtronic, Inc.	Enpulse E2DR01	For rapid RV pacing
CRT pulse generator	St. Jude Medical	Anthem PM 3212	For CRT performance
Multi-channel electrophysiologic recorder	GE Medical Systems	2003232-004	For surface and intracardiac electrogram
Catheter input module	GE Medical Systems	301-00202-08	Multiple pole switches for stimulation or recording
Radiofrequency generator	Johnson-Johnson Company	ST-4460	For RF current delivery
Cordless return electrode	Covidien	E7509	For current circuit formation
Cordis 6-Fr sheath	Johnson-Johnson Company	504-606X	Access for mapping catheter
Cordis 7-Fr sheath	Johnson-Johnson Company	504-607X	Access for mapping and ablation catheter
6-Fr quadripolar catheter	Johnson-Johnson Company	F6QRA005RT	Mapping catheter
7-Fr 4mm-tip steerable ablation catheter	St. Jude Medical	402823	Mapping and ablation catheter
Prucka Cardio-Lab®2000	GE Medical Systems	6.9.00.000	Software package for electrogram recording
Heparin	Haitong Pharmaceutical Co., Ltd	160505	Anticoagulant during catheter ablation
Digital image analysis system	Leica Microsystems	Qwin V3	For histologic analysis